BIAOAVAIBALITY & BIOEQUIVALENCE

Bioavailability:
It is rate and extent of absorption of unchanged drug from its dosage form.
For examples: Rate (time or rapidity) - acute conditions- asthma, pain etc.
Extent (amount) – chronic conditions- hypertension.
If the size of the dose to be administered is same, then bioavailability of a drug from its dosage form depends
upon 3 major factors –

1} Pharmaceutical factors
2} Patent related factors
3} Route of administration
Influence of route of administration
PARENTRAL> ORAL> RECTAL>TOPICAL ------- with few exceptions

Systemic availability: - The amount of drug that reaches the systemic circulation is called as Systemic availability
or simply Availability. It is denoted by F.
F = Bioavailable dose / Administered dose

Objective of Bioavailable studies:

 Development of a dosage form for a new drug entity.
 Determination of influence of excipients & patient related factors on the efficacy of absorption.
 Development of new formulations of the exciting drugs.
 Control of quality of a drug product during the early stages of marketing, in order to determine the influence of
processing factors, storage and stability on drug absorption.

CONSIDERATIONS OF BIOAVAIABILITY STUDY DESIGN

1. Bioavaibality: (Absolute v/s relative)
Absolute bioavailability:
When systemic availability of a drug administered orally is determined in comparison to its I.V. administration.
It is denoted by F.
Relative bioavailability:
When systemic availability of a drug after oral administration is compared with that of oral standard of the
same drug (Solution or suspension) and denoted by Fr.
2. Single dose v/s multiple dose studies
Single dose bioavailability studies are very common, easy, less exposure to drugs, less tedious. However it is
difficult to predict the steady state characteristics and inter subject variability with these studies.
Advantages of multiple dose study:
 Accurately reflects manner in which drug will be used clinically.
 Requires collection of few blood samples.
 Drug levels are higher due to cumulative effect and useful for less sensitive analytical methods
 Better evaluation of controlled released formulation.
 Nonlinearity if present, can be easily determined.
 No need of long wash out periods.
3. Human volunteer: (Healthy person v/s patient)
Generally bioavailability study should be carried out in patients, as patient get benefited from the study, reflects
better therapeutic efficacy, drug absorption pattern in disease state can be studied, avoids ethical quandary of
administering drug to healthy subjects.

BIOAVAIBILITY Mr. Dipak B. Bari (M. Pharm)

 But there are also various drawbacks like diseases, other drugs, physiological changes, fasting state is difficult
etc. Hence usually these studies performed on young (20-40 years old) healthy male adult volunteers (body
weight ± 10%) under restricted dietary and fixed activity conditions.

4. Methods of Bioavailability measurement

Pharmacokinetic methods
1. Plasma level time studies: Most reliable method of choice comparison to urine data method
A) Single dose: serial blood samples collection – 2-3 half-life.
Plot a graph concentration vs time.
B) For I.V. Sampling started within 5 min and subsequent samples at 15 min intervals.
C) For oral dose at least 3 points taken on absorption curve (ascending part)
Parameters considered important in plasma level time studies
 Cmax: It is peak plasma concentration. It increases with dose as well as increase in rate of absorption.
 Tmax: The peak time at which Cmax attended.
 AUC: Area under curve explains about amount of drug.

BIOAVAIBILITY Mr. Dipak B. Bari (M. Pharm)

 A) For single dose study:

B) For Multiple dose study:

C) For study state concentration:

2. Urinary excretion studies:

This method is based on the principle that the urinary excretion of unchanged drug is directly proportional to
the plasma concentration of drug.
It can be performed if
-At least 20% of administered dose is excreted unchanged in urine.
The study is useful for
 Drugs that extensively excreted unchanged in urine eg. Thiazide diuretics.
 Drugs that have urine as site of action eg. Urinary antiseptics like nitrofurontoin.

Steps involved:
Collection of urine at regular intervals for 7 half lifes.
Analysis of unchanged drug in collected sample.
Determination of amount of drug at each interval and cumulative as well.

Criteria's must be followed

At each sample collection total emptying of bladder is necessary.
Frequent sampling is essential in the beginning to compute correct rate of absorption.
The fraction excreted unchanged in urine must remain constant.

Parameters considered important in Urinary excretion studies

 (dXu/dt) max: Maximun urinary excretion rate
 (Tu) max: Time for maximum excretion rate
 Xu: Cumulative amount of drug excreted in the urine.

A) For single dose:

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 B) For Multiple dose study to study state concentration:

Pharmacodynamics methods:
1. Acute pharmacological response:
When bioavailability measurement by pharmacokinetic methods is difficult, inaccurate or non-reproducible
this method is used. Such as ECG, EEG, Pupil diameter etc.
It can be determined by dose response graphs. Responses measure for at least 3 half lifes.
Disadvantages:
 Pharmacological response is variable and accurate correlation drug and formulation is difficult.
 Observed response may be due to active metabolite.
2. Therapeutic response:
This method is based on observing clinical response in patients.
Drawbacks:
 Quantitation of observed response is too improper.
 The physiological status of subject assumed that does not change significantly over duration of study.
 If multiple dose protocols are not involved. Patient receive only single dose for few days or a week
 The patient’s receiving more than one drug treatment may be compromised due to drug-drug
interaction.

Drug Dissolution Rate & Bioavailability:

Dissolution Rate – Greatest influence on drug absorption.
In Vivo Bioavailability – best way to assess therapeutic efficacy – CPCSEA guidelines & cost.
Thus to assure batch to batch variations, inexpensive in vitro methods are used.
In vitro dissolution testing models:
The physicochemical property of most drugs that has greatest influence on absorption from GIT is dissolution rate.
However in vitro dissolution is good substitute for in vivo study in terms of saving cost and time. The best available
tool today which can at least quantitatively assure about the bioavailability of drug from its formulation is in vitro
dissolution test.
 Factors must be considered in the design of dissolution test are:
 Factors related to Dissolution Apparatus.
 Factors related to Dissolution Media/Fluid.
 Process Parameters for Dissolution Study.
Types of Dissolution Apparatus
 Closed Compartment – as per USP, 7 types
 Open Compartment
 Dialysis System
Biopharmaceutical Classification System for Drugs:

Class Solubility Permeability

Class І High High
Class ІІ Low High
Class ІІІ High Low
Class ІV Low low

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 In vitro- in vivo correlations (IVIVC):

It is defined as the predictive mathematical model that describes the relationship between in vitro property (rate &
extent of dissolution) and in vivo response (plasma drug concentration).

Objective:
The main objective of developing and evaluating IVIVC is to use dissolution test to serve as alternate for in vivo study
in human beings.
To ensure batch to batch consistency.
To serve as a tool for the development of new dosage form.
Approaches of IVIVC:
1. By establishing a relationship, usually linear, between the in vitro dissolution & the in vivo bioavailability
parameters.
2. By using the data from previous bioavailability studies to modify the dissolution methodology in order to
arrive meaningful IVIVC.
Correlations of IVIVC:
1. Correlations based on the plasma level data.
2. Correlations based on the urinary excretion data.
3. Correlations based on the pharmacologic response.
Levels IVIVC:
Level A: The highest category of correlation. A correlation of this type is generally linear and represents a point-to-
point relationship between in vitro dissolution and the in vivo input rate (e.g., the in vivo dissolution of the drug
from the dosage form)
Advantages: serves as alternate for in vivo study, change in manufacturing. Procedure or formula can be justified
without human studies.
Level B: The mean in vitro dissolution time is compare with mean in vivo residence time. It is not point to point
correlation. Data can be used for quality control standards. A Level B IVIVC uses the principles of statistical moment
analysis. The mean in vitro dissolution time is compared either to the mean residence time or to the mean in vivo
dissolution time. A Level B correlation does not uniquely reflect the actual in vivo plasma level curve, because a
number of different in vivo curves will produce similar mean residence time values.
Level C: It is single point correlation. e.g. t50%, Tmax, Cmax. This level is only useful as guide for formulation
development or quality control.
Level In vitro In vivo
A Dissolution curve Input (absorption) curves
B Statistical moments: MDT Statistical moments: MRT,
MAT, etc.
C Disintegration time,Time to have Cmax, Tmax, Ka, Time to have
10,50,90% dissolved, 10,50,90% absorbed,AUC
Dissolution rate, Dissolution efficiency (total or cumulative),
Table 1.Various parameters used in IVIVC depending on the level.

BIOAVAIBILITY Mr. Dipak B. Bari (M. Pharm)

 TYPES OF BIOEQUIVALENCE STUDIES:
For two products, pharmacokinetic equivalence (i.e. bioequivalence) is established if the rate and extent of
absorption of the active substance investigated under identical and appropriate experimental conditions only differ
within acceptable predefined limits.
Rate and extent of absorption are typically estimated by Cmax (peak concentration) and AUC (total exposure over
time), respectively, in plasma.
In vivo bioequivalence studies: (When needed)
1. Oral immediate release product with systemic action
 Indicated for serious conditions requiring assured response.
 Narrow therapeutic window.
 Complicated pharmacokinetic, absorption <70%, presystemic elimination>70%, nonlinear kinetics.
2. Non-oral immediate release products
3. Modified release products with systemic action.

Bioequivalence of two products can be assessed using in vitro standards, pharmacokinetic profile, clinical or
pharmacodynamic end points. Different approaches for determination of bioequivalence of a drug product are:
 An in vivo test in humans in which the concentration of the active ingredient and when appropriate, its
active metabolites, in blood, plasma, serum or other suitable biological fluid is measured as a function of
time.
 An in vivo test in humans in which the urinary excretion of the active ingredient and when appropriate, its
active metabolites are measured as a function of time.
 An in vitro test that has been correlated with and is predictive of human bioavailability profile or the one
acceptable to FDA (e.g. dissolution rate test) that ensures human in vivo bioavailability.
 An in vivo test in humans in which an appropriate pharmacological effect of the active ingredient and when
appropriate, its active metabolites are measured as a function of time if this effect can be measured with
adequate accuracy, sensitivity and reproducibility.
 Well-controlled clinical trials that establish the efficacy and safety of the drug product, for purpose of
determining bioavailability, or comparative clinical trials, for purpose of demonstrating bioequivalence.
 Any other approach considered adequate by the FDA to measure bioavailability or ascertain bioequivalence

In vitro bioequivalence studies: If none of the above criteria is applicable comparative in vitro dissolution studies
can be done.
Biowaivers: In vivo studies can be exempted under certain conditions.
1. Drug product only differ in strength of drug provided,
 Their pharmacokinetics are linear, Drug & excipient ratio is same,
 Bboth products manufactured by same manuf. at same site.
 Bioequivalence study done for original product, dissolution rate same under same conditions.
2. The method of production slightly modified in a way that not affect bioavailability.
3. The drug product meet following requirements: The product is in solubilised form, no excipients affecting
absorption, Topical use, Oral but not absorbed, inhalation as gas or vapour.

Bioequivalence experimental study designs:

1. Completely randomized designs:
All treatments are randomly allocated among all experimental subjects.
E.g. If there are 20 subjects, number from 1 to 20, randomly select non repeating numbers among these labels for
the first treatment. And then repeat for all other treatments.
Advantages:
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  Easy to construct.
 Can accommodate any number of treatment and subjects.
 Simple to analyze.
Disadvantages:
 Although can be used for number of treatments, but suited for few treatments.
 All subjects must be homogenous or random error will occur.

2. Randomized block designs:

First subjects are sorted in homogenous groups, called blocks and then treatments are assigned at random within
blocks.
Advantages:
 Systematic grouping gives more precise results.
 No need of equal sample size, any number of treatments can be followed, statistical analysis is simple, block
can be dropped and variability can be introduced.
Disadvantages:
 Missing observations in a block require more complex analysis.
 Degree of freedom is less.

3. Repeated measures, cross over designs:

It is a kind of randomized block design where same subject serves as a block. Same subject utilized repeatedly so
called as repeated measure design.
The administration of two or more treatments one after the other in a specified or random order to the same
group of patients is called cross-over designs.
Advantages:
 Good precision, Economic, can be performed with few subjects, useful in observing effects of treatment
over time in the same subject.
Disadvantages:
 Order effect due to position in treatment order.
 Cary over effect due to preceding treatment.
 Wash out period necessary – 10 elimination half lifes.

4. Latin square designs:

All other above designs are continuous trial. However in Latin square design each subject receives each treatment
during the experiment.
It is a two factor design (Rows=Subjects and Columns=Treatments). Carry –over effects are balanced.
Advantages:
 Minimize variability of plasma profiles and carry-over effects. Small
 Scale experiments can be carried out for pilot studies. Possible to focus on formulation variables.
Disadvantages:
 Less degree of freedom, randomization is complex, long time study, more formulations more complex
study, subject dropout rates are high.

Statistical interpretation of bioequivalence data:

After the data has been collected, statistical methods must be applied to determine the level of significance or any
observed difference in rate and /or extent of absorption to establish bioequivalence between two or more drug
products.
1. Analysis of varience (ANOVA): It is statistical procedure use to test data for differences within and between
treatment and control groups. A statistical difference between the pharmacokinetic parameters obtained from two

BIOAVAIBILITY Mr. Dipak B. Bari (M. Pharm)

or more drug products is considered statistically significant if there is probability of less than 1 in 20 or 0.05 (p≤0.05) .
The value of p indicates the level of statistical significance.

2. Confidence interval approach: It is also called as two one-sided procedure and used to demonstrate if
bioavailability of test product is too low or too high in comparison to reference product. 90% confidence interval of
two drug products must be within ±20% for bioavailability parameters such as AUC or Cmax. (i.e. between 80 to 102
%). For log transformed data 90% confidence interval is set at 80-125%.

Bioequivalence Studies:
Equivalence: - It is a relative term that compares drug product with respect to a specific characteristic or function or
to a defined set of standards.
1} Chemical equivalence: - When two or more drug products contain the same chemical substance as an active
ingredient in the same amount it is called chemical equivalence.
2} Pharmaceutical equivalence:- When two or more drug products are identical in strength, quality, purity, content
uniformity, disintegration, dissolution characteristics, they may however differ in containing different excipients,
called as pharmaceutical equivalence.
3} Bioequivalence:- It is relative term that denotes drug substance in two or more identical dosage forms, reaches the
systemic circulation at the same relative rate and to the same relative extent i.e. their plasma concentration-time
profiles will be identical without significant statistical differences.
When statistical differences are observed in the bioavailability of two or more drug products, Bioequivalence is
indicated.
4} Therapeutic Equivalence: - When two or more drug products that contain the same therapeutically active
ingredient, elicit identical pharmacologic effects and can control the disease to the same extent.

METHODS FOR ENHANCEMENT OF BIOAVAILABILITY

Approaches for enhancement of bioavailability –
The pharmaceutical approach.
The pharmacokinetic approach.
The biological approach.

METHODS:
1. Micronization
2. Use of surfactant
3. Use of salt forms
4. Alteration of pH of the drug microenvironment
5. Use of metastable polymorphs
6. Solute – solvent complexation
7. Solvent deposition
8. Selective absorption on insoluble carrier
9. Solid solution
 Use of solid solution
 Use of eutectic mixture
 Use of solid dispersion
10. Molecular encapsulation with cyclodextrins
11. Use of Co solvents.
12. Hydrotropy.

BIOAVAIBILITY Mr. Dipak B. Bari (M. Pharm)

 1. Micronization.
The particle size reduction technique enhance the solubility and dissolution rate of poorly water soluble drugs due
to the enormous surface that is generated. The process involves reducing the size of the solid drug particle to 1 to
10 microns commonly by spray drying or by use of air attrition methods such as fluid energy mill, jet mill, rotor stator
colloid mill etc. The process is also called as “Micromilling”. Micronization is not suitable for drugs having a high
dose number because it does not change the saturation solubility of the drug. Micronization of drug is not preferred
because micronized product has the tendency of agglomeration, which leads to decreased effective surface area for
dissolution. Figure 2 depicts the increase in window of absorption of a drug by Micronization.
Methods:
Spray drying
Air attrition methods.
E.g. : Aspirin, Griseofulvin, Steroidal compounds & Sulfa drugs.

2. Use of surfactants.
‘Surfactants promote wetting & penetration of fluids into solid drug particles.’
Surfactants are amphiphilic in nature having a polar end (the circular head) and non-polar end (the tail). When a
surfactant such as tween-80, sodium lauryl sulphate is placed in water, it will form micelles. A non-polar drug will
partition into the hydrophobic core of the micelle and the polar tail will solubilize the complex. This has been
illustrated by solubilization and wetting effects of bile salts on the dissolution of steroids
E.g. Spironolactone

3. Use of salt forms.

E.g. Alkali metal salts of acidic drugs like penicillins.
Strong Acid salt of basic drugs like atropine.

4. Alteration of pH of drug microenvironment.

Insitu salt formation
Buffered formulation e.g. Aspirin
The pH of solvent when reduced causes solubility enhancement. A combined effect of pH and complexation on
solubilization is also synergistic in nature. It was attempted to enhance dissolution of gliclazide using pH change
approach

5. Use of Metastable Polymorphs.

More stable than stable polymorph
E.g. Chloramphenicol palmitate B.

6. Solute-solvent complexation:
Solvates of drugs with organic solvents (pseudo polymorphs) have higher aqueous solubility than their respective
hydrates or original drug.
E.g. 1:2 Griseofulvin – Benzene solvate.

7. Selective adsorption on insoluble carriers.

A highly active adsorbent like inorganic clay e.g. Bentonite, enhance dissolution rate by maintaining concentration
gradient at its maximum. In this method, the poorly aqueous soluble drugs is dissolved in an organic solvent like
alcohol and deposited on an inert, hydrophilic, solid matrix such as starch or microcrystalline cellulose followed by
evaporation of solvent19 . Enhancement of dissolution rate of piroxicam using liquisolid compacts is an example
illustrating this technology20. The dissolution rate of a poorly soluble drug indomethacin was enhanced using
liquisolid compacts.
BIOAVAIBILITY Mr. Dipak B. Bari (M. Pharm)
 E.g. Griseofulvin, Indomethacin & Prednisone.

8. Eutectic mixture.
It is intimately blended physical mixture of two crystalline components.
Paracetamol –urea, Griseofulvin – urea & Griseofulvin-succinic acid
Disadvantage:
Not useful in:
 Drugs which fail to crystallize from mixed melt.
 Thermo labile drugs
 Carrier like succinic acid decompose at their melting point.

9. Molecular encapsulation with Cyclodextrins.

Considerable increase in solubility and dissolution of the drug has been achieved by the use of cyclodextrins. These
complexes can be prepared with ß-cyclodextrin (ß-CD) and HP-ß-CD; the required quantity of ß-CD is weighed and
water added to get tough consistency. To the mass, weighed quantity of the drug is added. The mixture is kneaded
in a glass mortar for one hour and then completely dried in hot air oven at 60 0C for 2 hours. The dried mass is
sieved through mesh no.120.
These molecules have inside hydrophobic cavity to accommodate lipophilic drug, outside is hydrophilic.
E.g. Thiazide diuretics, Barbiturates, Benzodiazepines & NSAIDS.

10. Use of cosolvents.

The addition of a water-miscible or partially miscible organic solvent is a common and an effective way to increase
the solubility of a non-polar drug. This process is known as cosolvency and the solvents used in combination to
increase the solubility of the drugs are known as cosolvents. The cosolvent system works by reducing the interfacial
tension between the predominately aqueous solution and the hydrophobic solute. It is also commonly referred to
as solvent blending. Co solvents such as ethanol, propylene glycol, glycerin, sorbitol and polyoxyethylene glycols can
be used. Ternary diagrams are used to visualize where maximum solubility occurs when more than one solvent is
used.

11. Hydrotropy.
Hydrotropy is a solubilization process whereby addition of large amounts of a second solute (Hydrotropic agents)
results in an increase in the aqueous solubility of another solute. Solute consists of alkali metal salts of various
organic acids. Hydrotropic agents are ionic organic salts. Additives or salts that increase solubility in given solvent
are said to “salt in” the solute and those salts that decrease solubility “salt out” the solute. Several salts with large
anions or cations that are themselves very soluble in water results in “salting in” of non-electrolytes called
“hydrotropic salts” a phenomenon known as “Hydrotropism”. The solubility of rofecoxib was enhanced by using
hydrotropes such as urea and nicotinamide
THANK YOU

BIOAVAIBILITY Mr. Dipak B. Bari (M. Pharm)